(CNF) as Scaffold Material - American Chemical Society

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Chapter 9

Tissue Engineering Using Plant-Derived Cellulose Nanofibrils (CNF) as Scaffold Material Kristin Syverud* 1PFI

Nanocellulose and Carbohydrate Polymers, Høgskoleringen 6b, 7491 Trondheim, Norway 2NTNU, Department of Chemical Engineering, 7491 Trondheim, Norway *E-mail: [email protected].

This chapter focuses on the use of cellulose nanofibrils (CNF) as scaffold material in tissue engineering. Tissue engineering is regeneration of human tissue by a combination of stem cells, a scaffold and growth factors. The field requires an interdisciplinary approach with expertise from multiple fields including medicine, biology, chemistry, material science and engineering. Knowledge about cell behavior is crucial for being successful in design of scaffolds. In this book chapter, central aspects of tissue engineering are pointed out, in particular requirements for scaffolds. Biocompatibility, surface chemistry, mechanical strength, porosity and controlled material degradation are all properties that are important for scaffold functionality. Focus is set on how this can be complied within the engineering of CNF based scaffolds.

Introduction Cellulose is the structural element in green plants, produced from glucose synthesized by the photosynthesis and that all life on earth relies on. Cellulose exists in nature always as nanoscaled microfibrils - never as single molecules that are arranged in larger fibril aggregates and fibre structures. The stiffness of cellulose fibrils is a property that makes this material different from many others and of particular interest in material research. In tissue engineering, this is one © 2017 American Chemical Society Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


of several properties that make cellulose nanofibrils a promising candidate as scaffold material. Cellulose is non-toxic, and cellulose nanofibril (CNF) forms hydrogels with high water content. This is useful for controlled drug release, wound dressings and in tissue engineering. The surface is hydrophilic having numerous hydroxyl groups that are also suitable sites for chemical modifications. The fibrils themselves are stiff, and fibril structures with adjustable stiffness can be formed in various ways. By combinations with other polymers, the combined material properties can be tailored. Hydrogels can also be transformed into porous structures which can be useful in construction of tissue scaffolds. Nanocellulose is often used as a common term for isolated cellulosic materials with dimensions in the nanometer range, and comprises bacterial nanocellulose (BNC), cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). In this book chapter, the main focus is on CNF, although there are several studies on both BNC and CNC in tissue engineering as well. There are now many well-known routes to produce CNF giving different surface chemistries. For example, functional groups can be introduced by TEMPO mediated oxidation giving aldehyde and carboxylic acid groups in the C6 position (1), mild carboxymethylation gives carboxymethyl groups (2) and periodate oxidation introducing aldehyde groups in position C2 and C3 of cellulose (3). Tissue engineering is regeneration of missing or damaged tissue by combining cells from the patient itself, a scaffold that the cells can adhere to and that can guide tissue formation, and nutrients and growth factors for the cells. The dimensions of the relevant cells are typically from 10 to 100 µm. Thus, interacting with cells requires working in microscale. In the human body, we have more than 200 types of cells that form our tissues and organs. Among the cells, stem cells are self-renewal type and can transform themselves into different specialized cells. Stem cells are used in tissue engineering. There are different types of stem cells, and they are categorized according to their differentiation potential, for example can multipotent stem cells differentiate into some tissue types, but not all. One example is mesenchymal stem cells that can differentiate into bones, muscles, cartilage and adipose cells. The behavior of the stem cells is not only dependent on the genetic information they carry, but is also regulated by their micro-environment. This is the extracellular matrix (ECM), which provides structural and biochemical support for the surrounding cells (4). ECM is a non-cellular three-dimensional macromolecular network. The major constituents of ECM are fibrous-forming proteins like collagen, elastin, fibronectin, laminins, glycoproteins, glycosaminoglycans (GAGs) and others. The structure is highly hydrated. Cells interact with the ECM through surface receptors. All cell types synthesize and secrete matrix macromolecules, and are thus participating in the formation of ECM. The ECM is composed of a large number of different molecules and is very complex. The precise composition and structure vary from tissue to tissue (5). It is thus not one ECM, but several tissue-specific ECMs. Through research, specific matrix properties that regulate cell adhesion and function have been identified. ECMs are hydrogels with anisotropic fibrillar architecture. Natural ECMs modulate tissue dynamics through their ability to bind and release bioactive molecules. It is a flow of information between cells and their ECM, and this is bidirectional (6). The 172 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


cells get their nutrients and oxygen from the thin walled capillaries of the cardiovascular system. In exchange, waste products pass in the reverse direction and are carried away with the bloodstream. Between the capillary and the cells is tissue fluid or extracellular fluid, in which the nutrients and oxygen is transported by diffusion. The capillary density is approximately 100 – 200 µm in highly metabolic tissue. The physical and chemical properties of the ECM, such as Young’s modulus, porosity, viscoelasticity, diffusivity and surface roughness are important for cell behavior (7). Tissue engineering is much about mimicking the ECM. The ECM is far too complicated and complex to be replicated by just using combinations of the various molecules it is composed of. Cooperation between cells, scaffold, growth factors and signaling molecules is required. The scaffold shall help the cells and guide them so that they can start the process of making an extracellular matrix themselves. An artificial or mimicked ECM should be adjustable to a particular biological environment to obtain cell- and tissue specificity. The scaffold shall support and guide three-dimensional tissue formation. To obtain this the scaffold should fulfil several functions; allow for cell attachment and migration, enable diffusion of cell nutrients and waste products, and have the right mechanical support for the cells (8). The scaffold should have a porous structure adequate for cells and with connective pores that permit growth of cells through the structure, and that allows for vascularization. Ideally, the scaffold should also be degradable and be absorbed by the surrounding tissue as the new tissue is formed. One obvious choice of scaffold material is collagen that constitutes a major part of the extracellular matrix in the body of humans. Collagen is a family of proteins comprising around 20 different types. They are responsible for tissue tensile strength and are both non-extensive and incompressible. Collagen is widely used in medicine, but has an inherent immunogenicity. Collagen carries the risk of being recognized as foreign by the patient’s immune system (9). Cellulose is a promising alternative in tissue engineering due to the non-toxicity, fibrous character, fibril stiffness, surface chemistry, gel formation and capability to form porous structures. If cellulose is fully degraded, the decomposition product is glucose, which is used as energy by the cells; an issue that will be discussed later. In the following, different properties regulating the interplay between scaffolds and cells are discussed with focus on how CNF scaffolds can be designed to meet the requirements for successful functionality.

Safety Aspects Natural cellulose is generally accepted as safe. Humans have always used cellulose, for example cellulose is a significant component in cereal products and vegetables that we are eating. When industrial production and use of the nanoscaled fibrils and crystals became a reality, the question about safety arose. It is known that cytotoxicity of a nanomaterial can be different from the cytotoxicity of their large scaled counterparts (10). Thus, several studies have been done on various types of nanocelluloses. Cytotoxicity tests of CNC using several different cell types showed no harmful effects in the tested concentration ranges 173 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


(11, 12). Suspensions of mechanically produced CNF at different concentrations were studied by Vartiainen et al. (2011) (13) using mouse macrophages and human monocyte derived macrophages. Macrophages are specialized cells in the forefront of the immune system, mainly located in the skin and lungs. The viability and cytokine profile of the cells were studied, and no cytotoxic effects were observed after 6 and 24 hours exposure. Cytotoxicity tests were also done using both direct and indirect contact between fibroblast cells and two types of CNF; unmodified CNF and CNF produced using TEMPO mediated oxidation (TO-CNF) as pre-treatment. For both these cellulosic materials, no harmful effects were observed on cell membrane, cell mitochondrial activity and DNA proliferation. Samples crosslinked using polyethyleneimine had however a significant reduction in cell viability (14). Figure 1 shows fibroblast cells in contact with CNF structures. Thus, initial cytotoxicity tests did not show harmful effects, as expected. This opened up, for investigations, use of CNF in biomedical applications, and more thorough cell studies were done in this connection. Interactions between materials and cells are continuously in focus in tissue engineering. This will be treated in some more detail in the following section about surface chemistry.

Figure 1. Image of freeze-dried CNF in direct contact with fibroblast cells. The star (∗) indicates a border area where the cells adhere to the medium. The white arrows indicate air-bubbles which is a confirmation of cell activity. Reproduced with permission from ref. (14). Copyright 2013 Springer.

174 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Mechanical Properties Cells can sense stiffness, and it has been shown that both hydrogel stiffness and single fibril stiffness influence cell responses (15). Collagen fibrils are composed of five tropocollagen triple helices which assemble into collagen microfibrils. They aggregate into fibrils with diameter between 10 and 500 nm. The fibrils further assembly into larger fibres. Yang et al. (2007) (16) have determined Young’s modulus of bovine Achilles tendon (type I collagen) with diameter of 200 – 350 nm and length of 20 – 200 µm, and got 5 GPa for the natural collagen and 15 GPa for crosslinked collagen. This is in agreement with other literature values for Young’s modulus of collagen, 0.2 – 7.5 GPa (17). Reported values for cellulose fibrils are somewhat higher. The strength of TO-CNF prepared from wood is reported to be 2 - 3 GPa corresponding to Young’s modulus of 30-40 GPa (18). It is not straight forward to control single fibril stiffness as this is given by the crystalline structure of cellulose. There are however many ways to control the fibril network stiffness, which has proven to be important. Cells pull and push against their surrounding matrix, and can feel the resistance to deformation of their adjacent environment. In response to the resistance, biochemical activity is actuated (19, 20). This process is called mechanotransduction (21). It has been demonstrated how matrix elasticity of polyacrylamid gels influenced the development of stem cells into differentiated cell types. Mesenchymal stem cells showed specified lineage towards neurons, myoblasts and osteoblasts depending on the elastic modulus of the polyacrylamide gels that otherwise were similar (22). Such results show how important it is to control elastic modulus of the scaffold in tissue engineering. The elasticity of natural tissue varies significantly throughout the body from very soft tissue like brain (E ~ 0.2 – 1 kPa) via medium soft muscle tissue (E ~ 10 kPa) to the stiff osteoid which is the unmineralized, organic portion of the bone matrix, mainly type I collagen (E ~30 – 45 kPa) (21). Being able to control the stiffness of CNF scaffolds within this range is thus a goal. CNF forms reversible or physical gels at low solid content (approx. 0.5 wt% depending on charge density, fibrillation degree and fibril length). Such fibril dispersions are held together by fibril entanglement, ionic interactions and hydrogen bonds. The elasticity of the gels can easily be increased by increasing the solid content. It has been shown that the storage modulus follows a power-law relation with respect to solid content of the CNF dispersions. The exponential values were 2.4 (23), 2.6 (24), and 3 (25) for carboxymethylated CNF, mechanically produced CNF and CNF produced using enzymatic pre-treament, respectively. Gels that do not have covalent bonds between fibrils are denoted reversible gels, also known as weak or false gels. They are usually not robust, and can easily be destroyed by changes in the chemical environment or if they are subjected to mechanical forces. They can even be diluted away by adding more solvent or swell as a result of diffusion. Their stability can be increased by crosslinking using di- or trivalent ions for CNF with a significant amounts of surface charges. For carboxylated CNF, the storage moduli of gels are strongly related to the valence of metal cation and their binding strength with carboxylate 175 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


groups on the fibrils. The storage moduli of CNF-metal ion gels followed the order of Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+. The CNF gel (1.27 wt%, surface carboxylate content 1.3 mmol/g) increased the storage modulus (G’) from 0.002 kPa to 3.4 kPa and 32 kPa by replacing Na+ with either Ca2+ or Fe3+, i.e. more than 10 000 times (26). Permanent or chemical gels can be formed by covalently crosslinking the fibrils. This can be done by combining with other polymers. Crosslinking of carboxylated bacterial nanocellulose (BNC) with chitosan through carbodiimidemediated amide binding is one example (27). When using TEMPO mediated oxidation as pre-treatment in fibril production, a significant amount of aldehyde groups are formed in addition to the carboxylic acids. The aldehyde groups can be used to crosslink the fibrils and obtain permanent gel-structures. An example is a study where crosslinks were created between aldehyde groups on fibrils and two polymers; either polyethyleneimine (PEI) or poly N-isopropylacrylamide-coallylamine-co-methylenebisacrylamide (pNIPA) through a cryo-gelation process (28). In another study small crosslinker molecules were used to crosslink cellulose fibres (29). Aqueous triamino-1,3,5-triazine (melamine) solutions were used to crosslink fibres in lyocell fabrics through creation of Schiff-bases. A Schiff base is the double bond which is created between the nitrogen on a primary amine containing molecule and the carbon on an aldehyde containing molecule. The double bond can later be stabilized by reduction to a secondary amine linkage, using a reduction agent such as sodium borohydride (NaBH4) (29). Exactly the same principle was followed in crosslinking fibrils for controlling the elastic modulus of CNF gels. Aldehyde groups were introduced through the TEMPO catalyzed oxidation in the production process of CNF, and were utilized in crosslinking using diamines. Diamines are commonly used as crosslinking agents in biomedical research (30), and ethylenediamine (EDA) and hexamethylendiamine (HMDA), differing by the length of the carbon chain and hence also the spacing distance between the functional amino groups were chosen. By altering the length of the crosslinker molecules and by varying the concentration, it was demonstrated how the elastic modulus of CNF based hydrogels could be controlled (31). This can be seen in Figure 2 showing the elastic moduli for the gels crosslinked with EDA and HMDA. In this work 2-picoline-borane replaced sodium borohydride as reducing agent as this could be done in one-pot reaction (32). In Figure 3 gels with the EDA and HMDA crosslinkers are shown during assessment in a texture analyzer, and also the effect of reduction with 2-picoline-borane. The brownish colour of the Schiff bases is then changed to white. The charge density of the fibrils will also influence the stiffness of the CNF matrix and hence their efficacy as scaffolds. Hydrogels based on TEMPO oxidized CNF having two different charge densities 1.14 ± 0.07 (low charge, LC) and 1.71 ± 0.04 mmol/g (high charge, HC) were compared with respect to proliferation of fibroblast cells. The hemicelluloses xylan (XYL), xyloglucan (XG) and galactoglucomannan (GGM) were added to the gels in various proportions using two methods, mixing CNF and hemicelluloses in predefined ratios, or in situ sorption by swelling CNF in a hemicellulose solution. Several gels were thus 176 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


prepared, half of them with the high charge. The cells did almost not grow in none of the high charged gels. The high surface charge could be the reason for not supporting cell survival. It could also be due to the low Young’s modulus (10 – 50 kPa) that accompanies the high charge when no crosslinking is done. In Figure 4, the interplay between cells and hydrogels are shown for the LC samples. The samples prepared by using in situ sorption of hemicellulose had very low cell proliferation (Figure 4 A and C). The composite hydrogels prepared by premixing the CNF and hemicelluloses, had in general higher stiffness compared to those with the in situ approach. Note the correlation between cell number per microscopic field and Young’s modulus of the corresponding gels (Figure 4 A) (33).

Figure 2. Young’s modulus (E) as a function of crosslinker concentration. Seven gels were prepared and measured for each crosslinker at each concentration. One series was made with crosslinker (HMDA) and 2-picoline-borane. The error bars show ± one standard deviation from the mean. Notice the reproducibility for the second replicates of H50 (H50 corresponds to 3.046 mmol HMDA per g CNF, E50 corresponds to 3.042 mmol EDA per g CNF) which were gels prepared independently from the first series. Reproduced with permission from ref. (31). Copyright 2015 Springer.

177 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. Gels with different crosslinkers during compression measurements with texture analyzer. From left to right: EDA, HMDA and HMDA with 2-picoline-borane. Reproduced with permission from ref. (31). Copyright 2015 Springer.

Structure and Porosity Cellulose nanofibrils are produced as nanoscaled fibrils dispersed in water. Depending on the production method, the fibril morphology (diameter, diameter distribution, length) can vary. Significant amounts of larger fiber fragments are usually also present (34). It is possible to control the fibril morphology by appropriate choice of production procedure. Thinner and more uniformly sized fibrils can be obtained when using chemical pre-treatments, e.g. TEMPO catalyzed oxidation to charge densities above 1 mmol/g cellulose. Less large fibre fragment can be obtained by increasing the amount of energy used in the fibrillation. Thus, it is possible to control fibril morphology to a large extent, although not completely independently from the surface chemistry, as surface chemistry also will change when a chemical pre-treatment is done. The morphology in the natural extracellular matrix varies also from nanometers to micrometers (5), indicating that variations in fibril morphology are not critical. The fibril dispersion can be utilized as it is, with or without ionic or covalent bonds between the fibrils; a network of entangled fibrils dispersed in an aqueous solution. This structure can also be changed into solid and highly porous structures by cryogelation or freeze-drying. In cryogelation, the dispersion is cooled down to temperatures below the freezing point, but above the eutectic point of the whole system. Ice crystals are thus formed, and all solid particles (fibrils) and solute are expelled into the liquid phase where a gelation occurs (35). Depending on the composition of the dispersion, the gelation can occur through covalent bonds, polymerization or non-covalent interactions like hydrogen bonds. If the interactions maintain after thawing, a porous material is formed. In freeze-drying the water is sublimated directly from the frozen state. In this way porous structures with walls composed of CNF can be formed. Figure 5 and 6 show examples of CNF structures prepared using cryogelation and freeze-drying respectively. The surfaces of the walls have a nano-scaled topography due to the nanofibrils they are composed of, see Figure 5 B. It has been shown that nano-scaled roughness plays an important role in cell adhesion and proliferation (36). 178 Agarwal et al.; Nanocelluloses: Their Preparation, Properties, and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 4. 3T3 cells were incubated with CNF matrices (charge density 1.14 mmol/g, denoted low charge, LC) in a density of (A) 3x105 cells/24-well or (C) 5×105 cells /24-well for 24 hours. (A and C): Proliferation of 3T3 cells in the CNF composite hydrogels and the Young’s modulus of the hydrogels. ± s.e.m.; n=5. **, p

(CNF) as Scaffold Material - American Chemical Society

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